Piconewton Forces Research Articles

Parsing digital or analog TCR performance through piconewton forces.

αβ T cell receptors (TCRs) principally recognize aberrant peptides bound to major histocompatibility complex molecules (pMHCs) on unhealthy cells, amplifying specificity and sensitivity through physical load placed on the TCR-pMHC bond during immunosurveillance. To understand this mechanobiology, TCRs stimulated by abundantly and sparsely arrayed epitopes (NP366-374/Db and PA224-233/Db, respectively) following in vivo influenza A virus infection were studied with optical tweezers. While certain NP repertoire CD8 T lymphocytes require many ligands for activation, others are digital, needing just few. Conversely, all PA TCRs perform digitally, exhibiting pronounced bond lifetime increases through sustained, energizing volleys of structural transitioning. Optimal digital performance is superior in vivo, correlating with ERK phosphorylation, CD3 loss, and activation marker up-regulation in vitro. Given neoantigen array paucity, digital TCRs are likely critical for immunotherapies.

In-Silico Analyses of Molecular Force Sensors for Mechanical Characterization of Biological Systems.

Mechanical forces play key roles in biological processes such as cell migration and sensory perception. In recent years molecular force sensors have been developed as tools for in situ force measurements. Here we use all-atom steered molecular dynamics simulations to predict and study the relationship between design parameters and mechanical properties for three types of molecular force sensors commonly used in cellular biological research: two peptide- and one DNA-based. The peptide-based sensors consist of a pair of fluorescent proteins, which can undergo Förster resonance energy transfer (FRET), linked by spider silk (GPGGA)n or synthetic (GGSGGS)n disordered regions. The DNA-based sensor consists of two fluorophore-labeled strands of DNA that can be unzipped or sheared upon force application with a FRET signal as readout of dissociation. We simulated nine sensors, three of each kind. After equilibration, flexible peptide linkers of three different lengths were stretched by applying forces to their N- and C-terminal Cα atoms in opposite directions. Similarly, we equilibrated a DNA-based sensor and pulled on the phosphate atom of the terminal guanine of one strand and a selected phosphate atom on the other strand in the opposite direction. These simulations were performed at constant velocity (0.01 nm/ns - 10 nm/ns) and constant force (10 pN - 500 pN) for all versions of the sensors. Our results show how the force response of these sensors depends on their length, sequence, configuration and loading rate. Mechanistic insights gained from simulations analyses indicate that interpretation of experimental results should consider the influence of transient formation of secondary structure in peptide-based sensors and of overstretching in DNA-based sensors. These predictions can guide optimal fluorophore choice and facilitate the rational design of new sensors for use in protein, DNA, hybrid systems, and molecular devices.

Direct investigations of the effects of nicardipine on calcium channels of astrocytes by Atomic Force Microscopy

Calcium channel blockers (CCB) of astrocytes can blockade the calcium ions entry through the voltage gated calcium channels (VGCC), and is widely used in the diseases related with VGCC of astrocytes. But many aspects of the interaction mechanisms between the CCB and VGCC of astrocytes still remain unclear due to the limited resolution of the approaches. Herein the effects of the nicardipine (a type of CCB) on VGCC of astrocytes were investigated at very high spatial, force and electrical resolution by multiple modes of Atomic Force Microscopy (AFM) directly. The results reveal that after the addition of nicardipine, the recognition signals of VGCC disappeared; the specific unbinding forces vanished; the conductivity of the astrocytes decreased (the current decreased about 2.9 pA and the capacitance was doubled); the surface potential of the astrocytes reduced about 14.2 mV. The results of electrical properties investigations are consistent with the simulation experiments. The relations between these biophysical and biochemical properties of VGCC have been discussed. All these demonstrate that the interactions between nicardipine and VGCC have been studied at nanometer spatial resolution, at picoNewton force resolution and very high electrical signal resolution (pA in current, pF in capacitance and 0.1 mV in surface potential) level. The approaches are considered to be high resolution and high sensitivity, and will be helpful and useful in the further investigations of the effects of other types of CCB on ion channels, and will also be helpful in the investigations of mechanisms and therapy of ion channelopathies.

Mechanical stimulation and electrophysiological monitoring at subcellular resolution reveals differential mechanosensation of neurons within networks

A growing consensus that the brain is a mechanosensitive organ is driving the need for tools that mechanically stimulate and simultaneously record the electrophysiological response of neurons within neuronal networks. Here we introduce a synchronized combination of atomic force microscopy, high-density microelectrode array and fluorescence microscopy to monitor neuronal networks and to mechanically characterize and stimulate individual neurons at piconewton force sensitivity and nanometre precision while monitoring their electrophysiological activity at subcellular spatial and millisecond temporal resolution. No correlation is found between mechanical stiffness and electrophysiological activity of neuronal compartments. Furthermore, spontaneously active neurons show exceptional functional resilience to static mechanical compression of their soma. However, application of fast transient (∼500 ms) mechanical stimuli to the neuronal soma can evoke action potentials, which depend on the anchoring of neuronal membrane and actin cytoskeleton. Neurons show higher responsivity, including bursts of action potentials, to slower transient mechanical stimuli (∼60 s). Moreover, transient and repetitive application of the same compression modulates the neuronal firing rate. Seemingly, neuronal networks can differentiate and respond to specific characteristics of mechanical stimulation. Ultimately, the developed multiparametric tool opens the door to explore manifold nanomechanobiological responses of neuronal systems and new ways of mechanical control.

Unravelling DNA Organization with Single-Molecule Force Spectroscopy Using Magnetic Tweezers.

Genomes carry the genetic blueprint of all living organisms. Their organization requires strong condensation as well as carefully regulated accessibility to specific genes for proper functioning of their hosts. The study of the structure and dynamics of the proteins that organize the genome has benefited tremendously from the development of single-molecule force spectroscopy techniques that allow for real-time, nanometer accuracy measurements of the compaction of DNA and manipulation with pico-Newton scale forces. Magnetic tweezers, in particular, have the unique ability to complement such force spectroscopy with the control over the linking number of the DNA molecule, which plays an important role when DNA-organizing proteins form or release wraps, loops, and bends in DNA. Here, we describe all the necessary steps to prepare DNA substrates for magnetic tweezers experiments, assemble flow cells, tether DNA to a magnetic bead inside a flow cell, and manipulate and record the extension of such DNA tethers. Furthermore, we explain how mechanical parameters of nucleoprotein filaments can be extracted from the data.

Force-Triggered Self-Destructive Hydrogels.

Self-destructive polymers (SDPs) are defined as a class of smart polymers that autonomously degrade upon experiencing an external trigger, such as a chemical cue or optical excitation. Because SDPs release the materials trapped inside the network upon degradation, they have potential applications in drug delivery and analytical sensing. However, no known SDPs that respond to external mechanical forces have been reported, as it is fundamentally challenging to create mechano-sensitivity in general and especially so for force levels below those required for classical force-induced bond scission. To address this challenge, the development of force-triggered SDPs composed of DNA crosslinked hydrogels doped with nucleases is described here. Externally applied piconewton forces selectively expose enzymatic cleavage sites within the DNA crosslinks, resulting in rapid polymer self-degradation. The synthesis and the chemical and mechanical characterization of DNA crosslinked hydrogels, as well as the kinetics of force-triggered hydrolysis, are described. As a proof-of-concept, force-triggered and time-dependent rheological changes in the polymer as well as encapsulated nanoparticle release are demonstrated. Finally, that the kinetics of self-destruction are shown to be tuned as a function of nuclease concentration, incubation time, and thermodynamic stability of DNA crosslinkers.

DNA Origami-Based Single-Molecule Force Spectroscopy and Applications.

Over the last years, single-molecule force spectroscopy provided insights into the intricate connection between mechanical stimuli and biochemical signaling. The underlying molecular mechanisms were uncovered and explored using techniques such as atomic force microscopy and force spectroscopy using optical or magnetic tweezers. These experimental approaches are limited by thermal noise resulting from a physical connection of the studied biological system to the macroscopic world. To overcome this limitation, we recently introduced the DNA origami force clamp (FC) which is a freely diffusing nanodevice that generates piconewton forces on a DNA sequence of interest. Binding of a protein to the DNA under tension can be detected employing fluorescence resonance energy transfer (FRET) as a sensitive readout.This protocol introduces the reader to the working principles of the FC and provides instructions to design and generate a DNA origami FC customized for a protein of interest. Molecular cloning techniques are employed to modify, produce, and purify a custom DNA scaffold. A fluorescently labeled DNA suitable to detect protein binding via FRET is generated via enzymatic ligation of commercial DNA oligonucleotides. After thermal annealing of all components, the DNA origami FC is purified using agarose gel electrophoresis. The final section covers the interrogation of the FC using confocal single-molecule FRET measurements and subsequent data analysis to quantify the binding of a DNA-binding protein to its cognate recognition site under a range of forces. Using this approach, force-dependent DNA-protein interactions can be studied on the single-molecule level on thousands of molecules in a parallelized fashion.

Introduction to Optical Tweezers: Background, System Designs, and Applications.

Optical tweezers are a means to manipulate objects with light. With the technique, microscopically small objects can be held and steered, allowing for accurate measurement of the forces applied to these objects. Optical tweezers can typically obtain a nanometer spatial resolution, a picoNewton force resolution, and a millisecond time resolution, which makes the technique well suited for the study of biological processes from the single-cell down to the single-molecule level. In this chapter, we aim to provide an introduction to the use of optical tweezers for single-molecule analyses. We start from the basic principles and methodology involved in optical trapping, force calibration, and force measurements. Next, we describe the components of an optical tweezers setup and their experimental relevance. Finally, we will provide an overview of the broad applications in context of biological research, with the emphasis on the measurement modes, experimental assays, and possible combinations with fluorescence microscopy techniques.

Entry of microparticles into giant lipid vesicles by optical tweezers.

Entry of micro- or nanosized objects into cells or vesicles made of lipid membranes occurs in many processes such as entry of viruses into host cells, microplastics pollution, drug delivery, or biomedical imaging. Here we investigate the microparticle crossing of lipid membranes in giant unilamellar vesicles in the absence of strong binding interactions (e.g., streptavidin-biotin binding). In these conditions, we observe that organic and inorganic particles can always penetrate inside the vesicles provided an external piconewton force is applied and for relatively low membrane tensions. In the limit of vanishing adhesion, we identify the role of the membrane area reservoir and show that a force minimum exists when the particle size is comparable to the bendocapillary length.

Measuring pico-Newton Forces with Lipid Anchors as Force Sensors in Molecular Dynamics Simulations.

Binding forces between biomolecules are ubiquitous in nature but sometimes as weak as a few pico-Newtons (pN). In many cases, the binding partners are attached to biomembranes with the help of a lipid anchor. One important example are glycolipids that promote membrane adhesion through weak carbohydrate-carbohydrate binding between adjacent membranes. Here, we use molecular dynamics (MD) simulations to quantify the forces generated by bonds involving membrane-anchored molecules. We introduce a method in which the protrusion of the lipid anchors from the membrane acts as the force sensor. Our results with two different glycolipids reveal binding forces of up to 20 pN and corroborate the recent notion that carbohydrate-carbohydrate interactions are generic rather than specific.

Regulation of branched versus linear Arp2/3‐generated actin filaments

Activation of the Arp2/3 complex by VCA‐motif‐bearing actin nucleation‐promoting factors results in the formation of “daughter” actin filaments branching off the sides of pre‐existing “mother” filaments. Alternatively, when stimulated by SPIN90, Arp2/3 directly nucleates “linear” actin filaments. Uncovering the similarities and differences between these two mechanisms is fundamental to understanding how actin cytoskeleton dynamics are regulated. Here, analysis of individual filaments reveals that, unexpectedly, the VCA motifs of WASP, N‐WASP, and WASH destabilize existing branches, as well as SPIN90‐Arp2/3 at linear filament ends. Furthermore, branch stabilizer cortactin and destabilizer GMF each have a similar impact on SPIN90‐activated Arp2/3. However, unlike branch junctions, SPIN90‐Arp2/3 at the ends of linear filaments is not destabilized by piconewton forces and does not become less stable with time. It thus appears that linear and branched Arp2/3‐generated filaments respond similarly to the regulatory proteins we have tested, albeit with some differences, but significantly differ in their responses to aging and mechanical stress. These kinetic differences likely reflect the small conformational differences recently reported between Arp2/3 in branch junctions and linear filaments and suggest that their turnover in cells may be differently regulated.

In-series vs. in-parallel: The role of cargo-motor connectivity in transport by ensembles of cytoskeletal motor proteins.

The movement of the cytoskeletal molecular motor proteins dynein and kinesin on microtubule networks facilitates the complex intracellular organization of eukaryotic cells. Ensembles of these motors are a prevalent feature of cytoskeletal transport, indicating possible synergistic effects of their use for intracellular organization. While the force output of individual motors is well studied, the mechanisms governing emergent force generated by multiple motors acting as an ensemble remains unclear. Determining the fundamental mechanisms controlling the interaction and integration of the forces produced by these motors at the nanoscale is essential to understanding the broader system of intracellular transport. Using total internal reflection fluorescence (TIRF) microscopy, we are investigating the role of the spatial arrangement of motors in an ensemble with a particular emphasis on the connectivity between motors and their cargo. Using motors purified from yeast and the nanoscale construction techniques of DNA origami, we create programmable chassis structures in which multiple cytoplasmic dyneins or Kip2 kinesins are arranged with independent, “in parallel” connections to a resistive cargo load or, in contrast, are arranged with a single, “in series” shared cargo connection. We expect the use of these chassis will allow us to determine the mechanisms behind the structural integration of the piconewton forces produced by cytoskeletal molecular motors within ensembles, ultimately providing insight on how the individual connectivity of motors to one another and their cargo affects load-sharing, force integration, and the resultant emergent motility of the motor ensemble.

Tubulins show susceptibility to magnetic nanoparticle interaction.

Low piconewton forces can induce axonal specification and outgrowth in neurons. However, the secondary mechanism underlying force-mediated axon specification is poorly understood. Within the axon shaft, microtubules, composed of α and β tubulin dimers, are stabilized by the protein Tau. These microtubules polymerize toward the axonal tip, where actin provides a tensile force against the membrane to maintain or extend the structure of the tip. We hypothesize that exogenous forces manipulate one or more of these structural proteins, which could induce axonal lineage. Our previous work has shown that hTau40, the brain's most ubiquitous isoform of Tau, can be directly dragged by internalized magnetic nanoparticles (MNPs) within primary neurons. Here, we sought to establish whether this direct relationship between the nanoparticles and Tau applied to other axonal structural proteins. We transduced (BacMam) dissociated primary cortical neurons (rat, E18) at the day in vitro two (DIV 2) with one of three vectors containing a gene for a fluorescently labeled structural protein: α-tubulin-mCherry, β-tubulin-GFP, or β-actin-GFP. At DIV 4, the neurons were incubated for 24 h with starch-NH2-coated, far-red fluorescent magnetic nanoparticles (d: 100 nm) at 10 µg/mL. Cells were then washed and imaged using a Leica DMi8 inverted fluorescent microscope to determine whether there was high colocalization, indicating direct interaction, between the MNPs and the fluorescent proteins. Compared to the high-intensity protein spots, α-tubulin-mCherry showed the greatest percentage of correlated MNP clusters (45.7 ± 6.8 %, n = 11 cells), followed by β-tubulin-GFP, with 36.9 ± 11.7 % (n = 6) of high protein spots correlated to MNP clusters. Only 8.27 ± 5.32% (n = 7) of β-actin-GFP spots correlating to MNP clusters. These preliminary data suggest tubulins may be more susceptible to direct nanomagnetic force manipulation (mean ± SEM).

Single piconewton forces regulate dissociation of the Latrophilin-3 gain domain.

Latrophilins are adhesion G-protein coupled receptors (aGPCRs) that have been implicated in multiple neuropsychiatric diseases and are necessary for excitatory synapse formation in specific regions of the brain. Despite the importance of latrophilins in neurodevelopment, the mechanisms involved in signaling by latrophilins, and aGPCRs in general, remain poorly understood. Latrophilins are autoproteolytically cleaved at a GAIN domain conserved across aGPCRs, and the two resulting latrophilin fragments remain stably (but non-covalently) associated on the cell membrane. In one prevalent hypothesis, dissociation of the latrophilin fragments, potentially in response to mechanical forces applied through transsynaptic ligand binding, drives a conformational change in the transmembrane domain to initiate signal transduction. However, the stability of the association between latrophilin fragments calls into question how and whether their dissociation can occur on biologically relevant timescales. Here, we present evidence that physiological levels of mechanical force substantially accelerate the rate of dissociation of the Latrophilin-3 (Lphn3) GAIN domain. Using magnetic tweezers, we directly apply mechanical force in the single-pN range to individual Lphn3 GAIN domains and demonstrate that these forces are sufficient to dissociate the GAIN domain on the seconds-to-minutes timescale. We further observe that the GAIN domain fragments can reversibly reassociate after force-mediated dissociation. Moreover, the distribution of measured Lphn3 dissociation times suggests that the fragments of the Lphn3 GAIN domain form a two-state slip bond, with a highly force-sensitive transition between strongly- and weakly-bound states. Our results support the idea that mechanical force may be a key regulator of latrophilin signaling in synapse formation and, more broadly, signaling by aGPCRs.

Dynamic interfaces for contact-time control of colloidal interactions.

Understanding pairwise interactions between colloidal particles out of equilibrium has a profound impact on dynamical processes such as colloidal self assembly. However, traditional colloidal interactions are effectively quasi-static on colloidal timescales and cannot be modulated out of equilibrium. A mechanism to dynamically tune the interactions during colloidal contacts can provide new avenues for self assembly and material design. In this work, we develop a framework based on polymer-coated colloids and demonstrate that in-plane surface mobility and mechanical relaxation of polymers at colloidal contact interfaces enable an effective, dynamic interaction. Combining analytical theory, simulations, and optical tweezer experiments, we demonstrate precise control of dynamic pair interactions over a range of pico-Newton forces and seconds timescales. Our model helps further the general understanding of out-of-equilibrium colloidal assemblies while providing extensive design freedom via interface modulation and nonequilibrium processing.

APLF and long non-coding RNA NIHCOLE promote stable DNA synapsis in non-homologous end joining

The synapsis of DNA ends is a critical step for the repair of double-strand breaks by non-homologous end joining (NHEJ). This is performed by a multicomponent protein complex assembled around Ku70-Ku80 heterodimers and regulated by accessory factors, including long non-coding RNAs, through poorly understood mechanisms. Here, we use magnetic tweezers to investigate the contributions of core NHEJ proteins and APLF and lncRNA NIHCOLE to DNA synapsis. APLF stabilizes DNA end bridging and, together with Ku70-Ku80, establishes a minimal complex that supports DNA synapsis for several minutes under piconewton forces. We find the C-terminal acidic region of APLF to be critical for bridging. NIHCOLE increases the dwell time of the synapses by Ku70-Ku80 and APLF. This effect is further enhanced by a small and structured RNA domain within NIHCOLE. We propose a model where Ku70-Ku80 can simultaneously bind DNA, APLF, and structured RNAs to promote the stable joining of DNA ends.

Interfacial Layer Breaker: A Violation of Stokes’ Law in High-Speed Atomic Force Microscope Flows

Structured water near surfaces is important in nonclassical crystallization, biomineralization, and restructuring of cellular membranes. In addition to equilibrium structures, studied by atomic force microscopy (AFM), high-speed AFM (H-S AFM) can now detect piconewton forces in microseconds. With increasing speeds and decreasing tip diameters, there is a danger that continuum water models will not hold, and molecular dynamic (MD) simulations would be needed for accurate predictions. MD simulations, however, can only evolve over tens of nanoseconds due to memory and computational efficiency/speed limitations, so new methods are needed to bridge the gap. Here, we report a hybrid, multiscale simulation method, which can bridge the size and time scale gaps to existing experiments. Structured water is studied between a moving silica AFM colloidal tip and a cleaved mica surface. The computational domain includes 1,472,766 atoms. To mimic the effect of long-range hydrodynamic forces occurring in water, when moving the AFM tip at speeds from 5 × 10-7 to 30 m/s, a hybrid multiscale method with local atomistic resolution is used, which serves as an effective open-domain boundary condition. The multiscale simulation is thus equivalent to using a macroscopically large computational domain with equilibrium boundary conditions. Quantification of the drag force shows the breaking of continuum behavior. Nonmonotonic dependence on both the tip speed and distance from the surface implies breaking of the hydration layer around the moving tip at time scales smaller than water cluster formation and strong water compressibility effects at the highest speeds.

Facile detection of mechanical forces across proteins in cells with STReTCh

SummaryNumerous proteins experience and respond to mechanical forces as an integral part of their cellular functions, but measuring these forces remains a practical challenge. Here, we present a compact, 11-kDa molecular tension sensor termed STReTCh (sensing tension by reactive tag characterization). Unlike existing genetically encoded tension sensors, STReTCh does not rely on experimentally demanding measurements based on Förster resonance energy transfer and is compatible with typical fix-and-stain protocols. Using a magnetic tweezers assay, we calibrate the STReTCh module and show that it responds to physiologically relevant, piconewton forces. As proof of concept, we use an extracellular STReTCh-based sensor to visualize cell-generated forces at integrin-based adhesion complexes. In addition, we incorporate STReTCh into vinculin, a cytoskeletal adaptor protein, and show that STReTCh reports on forces transmitted between the cytoskeleton and cellular adhesion complexes. These data illustrate the utility of STReTCh as a tool for visualizing molecular-scale forces in biological systems.

Exploring cell and tissue mechanics with optical tweezers.

Cellular and tissue biosystems emerge from the assembly of their constituent molecules and obtain a set of specific material properties. To measure these properties and understand how they influence cellular function is a central goal of mechanobiology. From a bottoms-up, physics or engineering point-of-view, such systems are a composition of basic mechanical elements. However, the sheer number and dynamic complexity of them, including active molecular machines and their emergent properties, makes it currently intractable to calculate how biosystems respond to forces. Because many diseases result from an aberrant mechanotransduction, it is thus essential to measure this response. Recent advances in the technology of optical tweezers have broadened their scope from single-molecule applications to measurements inside complex cellular environments, even within tissues and animals. Here, we summarize the basic optical trapping principles, implementations and calibration procedures that enable force measurements using optical tweezers directly inside cells of living animals, in combination with complementary techniques. We review their versatility to manipulate subcellular organelles and measure cellular frequency-dependent mechanics in the piconewton force range from microseconds to hours. As an outlook, we address future challenges to fully unlock the potential of optical tweezers for mechanobiology.

On non-Kolmogorov turbulence in blood flow and its possible role in mechanobiological stimulation

The study of turbulence in physiologic blood flow is important due to its strong relevance to endothelial mechanobiology and vascular disease. Recently, Saqr et al. (Sci Rep 10, 15,492, 2020) discovered non-Kolmogorov turbulence in physiologic blood flow in vivo, traced its origins to the Navier–Stokes equation and demonstrated some of its properties using chaos and hydrodynamic-stability theories. The present work extends these findings and investigates some inherent characteristics of non-Kolmogorov turbulence in monoharmonic and multiharmonic pulsatile flow under ideal physiologic conditions. The purpose of this work is to propose a conjecture for the origins for picoNewton forces that are known to regulate endothelial cells’ functions. The new conjecture relates these forces to physiologic momentum-viscous interactions in the near-wall region of the flow. Here, we used high-resolution large eddy simulation (HRLES) to study pulsatile incompressible flow in a straight pipe of L/D=20. The simulations presented Newtonian and Carreau–Yasuda fluid flows, at R{e}_{m}approx 250, each represented by one, two and three boundary harmonics. Comparison was established based on maintaining constant time-averaged mass flow rate in all simulations. First, we report the effect of primary harmonics on the global power budget using primitive variables in phase space. Second, we describe the non-Kolmogorov turbulence in frequency domain. Third, we investigate the near-wall coherent structures in time and space domains. Finally, we propose a new conjecture for the role of turbulence in endothelial cells’ mechanobiology. The proposed conjecture correlates near-wall turbulence to a force field of picoNewton scale, suggesting possible relevance to endothelial cells mechanobiology.